5974 Biochemistry 1991, 30, 5974-5985 Milostone, L., Barton, D. E., Francke, U., & Broadus, A. E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 597. McKee, R. L., Goldman, M. E., Caulfield, M. P., DeHaven, P. A,, Levy, J. J., Nutt, R. F., & Rosenblatt, M. (1988) Endocrinology 122, 3008. McKee, R. L., Caulfield, M. P., & Rosenblatt, M. (1990) Endocrinology 127, 76. Merrifield, R. B. (1969) Adv. Enzymol. Subj. Biochem. 32, 221. Mosberg, H. I., Hurst, R., Hruby, V. J., Gee, K., Yamamura, H. I., Galligan, J. J., & Burks, T. F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5871. Mosely, J. M., Kubota, M., Difenbachen-Jagger, H., Wet- tenhall, R. E. H., Kemp, B. E., Suva, L. J., Rodda, C. P., Ebeling, P. R., Hudson, P. J., Zajac, J. T., & Martin, T. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5048. Nissenson, R. A., Diep, D., & Strewler, G. J. (1988) J. Biol. Chem. 263, 12866. Nutt, R. F., Caulfield, M. P., Levy, J. J., Gibbons, S. W., Rosenblatt, M., & McKee, R. L. (1990) Endocrinology 127, 491. Pizurki, L., Rizzoli, R., Moseley, J., Martin, T. J., Caverzasio, J., & Bonjour, J.-P. (1988) Am. J. Physiol. 255, F957. Rabbani, S. A,, Mitchell, J., Roy, D. R., Hendy, G. N., & Goltzman, D. (1988) Endocrinology 123, 2709. Rodan, S. B., Insogna, K. L., Vignery, A. M., Stewart, A. F., Broadus, A. E., D'Sousa, S. M., Bertolini, D. R., Mundy, G. R., & Rodan, G. A. (1983) J. Clin. Invest. 72, 1511. Rosenblatt, M., Segre, G. V., Tyler, G. A., Shepard, G. L., Nussbaum, S. R., & Potts, J. T., Jr. (1980) Endocrinology 107, 545. Shigeno, C., Yamamoto, I., Kitamura, N., Noda, T., Lee, K., Sone, T., Shiomi, K., Ohtaka, A., Fujii, N., Yajima, H., & Konishi, J. (1988) J. Biol. Chem. 263, 18369. Stewart, A. F., Insogna, K. L., Goltzman, D., & Broadus, A. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1454. Stewart, A. F., Wu, T., Goumas, D., Burtis, W. J., & Broadus, A. E. (1987) Biochem. Biophys. Res. Commun. 146,672. Strewler, G. J., Williams, R. D., & Nissenson, R. A. (1983) J. Clin. Invest. 71, 769. Strewler, G. J., Stern, P. H., Jacobs, J. W., Eveloff, J., Klein, R. F., Leung, S. C., Rosenblatt, M., & Nissenson, R. A. (1987) J. Clin. Invest. 80, 1803. Suva, L. J., Winslow, G. A., Wettenhall, R. E. H., Hammonds, R. G., Moseley, J. M., Diefenbach-Jagger, H., Rodda, C. P., Kemp, B. E., Rodriguez, H., Chen, E. Y., Hudson, P. J., Martin, T. J., & Wood, W. I. (1987) Science 237, 893. Thiede, M. A., Strewler, G. J.; Nissenson, R. A., Rosenblatt, M., & Rodan, G. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4605. Veber, D. F., & Freidinger, R. M. (1985) Trends. Neurosci. 8, 392. Veber, D. F., Holly, F. W., Paleveda, W. J., Nutt, R. F., Bergstrand, S. J., Torchiana, M., Glitzer, M. S., Saperstein, R., & Hirschmann, R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2636. Veber, D. F., Saperstein, R., Nutt, R. F., Freidinger, R. M., Brady, S. F., Curley, P., Perlow, D. S., Paleveda, W. J., Colton, C. D., Zacchei, A. G., Tocco, D. J., Hoff, D. R., Vandlen, R. L., Gerich, J. E., Hall, L., Mandarino, L., Cordes, E. H., Anderson, P. S., & Hirschmann, R. (1984) Life Sci. 34, 137 1. Solvent Denaturation and Stabilization of Globular Proteins? Darwin 0. V. Alonso and Ken A. Dill**$ Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 Received June 15, 1990; Revised Manuscript Received January 22, 1991 ABSTRACT: Statistical thermodynamic theory has recently been developed to account for the stabilities of globular proteins. Here we extend that work to predict the effects of solvents on protein stability. Folding is assumed to be driven by solvophobic interactions and opposed by conformational entropy. The solvent dependence of the solvophobic interactions is taken from transfer experiments of Nozaki and Tanford on amino acids into aqueous solutions of urea or guanidine hydrochloride (GuHCl). On the basis of the assumption of two pathways involving collapse and formation of a core, the theory predicts that increasing denaturant should lead to a two-state denaturation transition (i.e., there is a stable state along each path separated by a free energy barrier). The denaturation midpoint is predicted to occur at higher concentrations of urea than of GuHCl. At neutral pH, the radius of the solvent-denatured state should be much smaller than for a random-flight chain and increase with either denaturant concentration or number of polar residues in the chain. A question of interest is whether free energies of folding should depend linearly on denaturant, as is often assumed. The free energy is predicted to be linear for urea but to have some small curvature for GuHCl. Predicted slopes and exposed areas of the unfolded states are found to be in generally good agreement with experiments. We also discuss stabilizing solvents and compare thermal with solvent de- naturation. R o t e i n s can be denatured in the presence of certain small-molecule solutes in high concentrations, Examples of 'We thank the NIH, the Pew Foundation, and the DARPA URI *Address correspondence to this author. !Present address: UCSF Laurel Heights Campus, 3333 California St., such solutes are urea and guanidine hydrochloride. Other solutes in high concentration stabilize proteins. Examples of these are sugars, glycerol, polyols, and poly(ethy1ene glycol) (Arakawa, 1982; Back, 1979; GeUo & Timasheff, 1981; Lee & Lee, 1981; Lee & Timasheff, 1981). One problem in un- derstanding the molecular mechanism of nonspecific solute action on protein stability has been the absence of a molecular program for financial support. Room 102, San Francisco, CA 941 18. 0006-2960/9 1 /0430-5974$02.50/0 0 199 1 American Chemical Society